Abstract
The importance of microRNAs (miRs) in control of gene expression is now clearly recognized. While individual microRNAs are thought to target hundreds of disparate mRNAs via imperfect base pairing, little is known about the characteristics of miR target sites. Here we show that the miRs can be aligned with empirically identified accessible sites in a target RNA (Cytokeratin 19, KRT), and that some of the aligned miRs functionally down‐regulate KRT expression post‐transcriptionally. We employed an RNase‐H‐based random library selection protocol to identify accessible sites in KRT RNA. We then aligned the Sanger Institute database collection of human miRs to KRT mRNA, and also aligned them using the web‐based MicroInspector program. Most miRs aligned with the accessible sites identified empirically; those not aligned with the empirically identified sites also functioned effectively in RNase‐H‐based assays. Similar results were obtained with a second target RNA (Mammoglobin). Transient transfection assays established that some of the miRs which aligned with KRT significantly down‐regulated it at the protein level, with no effect on RNA level. The functionally effective miRs aligned within the coding region of KRT, whereas a number of miRs which aligned with the 3′‐untranslated region did not produce down‐regulation. J. Cell. Biochem. 109: 509–518, 2010. © 2009 Wiley‐Liss, Inc.